There is a current need for new methods for the non-invasive diagnosis of a variety of diseases such as thromboembolic disease, atherosclerosis, infection and cancer. Radiopharmaceuticals comprised of gamma-ray emitting radionuclide labeled biologically active molecules can fulfill the need. The biologically active molecules serve to localize the radionuclides at the sites of disease and thus allow the sites to be visualized by gamma scintigraphy. The molecules can be either proteins, antibodies, antibody fragments, peptides or polypeptides, or peptidomimetics. The molecules interact with a receptor or binding site expressed at the sites of the disease or with a receptor or binding site on an endogenous blood component, such as platelets and leukocytes, that accumulate at the sites. This interaction results in selective localization of a percentage of the injected radiopharmaceutical while the remainder is cleared either through the renal or hepatobiliary systems. The localized radiopharmaceutical is then imaged externally using gamma scintigraphy. The relative rates of localization, clearance and radionuclidic decay determine the ease of visualization, often expressed as the target-to-background ratio. Frequently, only certain portions of the biologically active molecules bind to the receptors; these portions are termed the recognition sequences or units.
A number of radiopharmaceuticals comprised of radionuclide labeled proteins, antibodies or antibody fragments are under development, however, to date only one has been approved by the Food and Drug Administration. This sparse record results from a combination of factors that make developing these radiopharmaceuticals difficult, including problems with manufacturing and quality control, non-optimal sequestration and clearance rates, and the occurence of antigenic or allergic responses to the radiopharmaceuticals. These problems are mainly due to the macromolecular nature of the proteins, antibodies and antibody fragments. Their high molecular weight makes direct chemical synthesis impractical, therefore they must be synthesized by recombinant or cloning techniques that typically give low yields and require extensive isolation and purification procedures. Their molecular weight can slow their rates of localization and preclude their clearance by an active elimination mechanism via the kidneys or liver, resulting in prolonged retention in the circulation which causes a high background level during imaging. Also, the body's immune system tends to recognize more efficiently larger exogenous species.
The use of lower molecular weight peptides, polypeptides or peptidomimetics as the biologically active molecules obviates a number of these problems. These molecules can be synthesized directly using classical solution chemistry or by an automated peptide synthesizer. They can be formed in higher yields and require less complicated purification procedures. They tend to clear more rapidly from the circulation by an active elimination pathway resulting in a lower background in the images. They are also usually not immunogenic. The first radionuclide labeled polypeptide radiopharmaceutical has been recently approved by the Food and Drug Administration.
There are two general methods for labeling biologically active molecules with radionuclides for use as radiopharmaceuticals termed direct and indirect labeling. Direct labeling involves attaching the radionuclide to atoms on the biologically active molecule; while the indirect method involves attaching the radionuclide via a chelator. The chelator can either be attached to the biologically active molecule prior to reaction with the radionuclide or the radionuclide labeled chelator moiety can be attached to the biologically active molecule. Several recent reviews describe these labeling methods and are incorporated herein by reference: S. Jurisson et. al., Chem. Rev., 1993, 93, 1137; A. Verbruggen, Eur. J. Nuc. Med., 1990, 17, 346; and M. Derwanjee, Semin. Nuc. Med., 1990, 20, 5.
The use of hydrazines and hydrazides as chelators to modify proteins for labeling with radionuclides has been recently disclosed in Schwartz et. al., U.S. Pat. No. 5,206,370. The protein is modified by reaction with bifunctional aromatic hydrazines or hydrazides having a protein reactive substituent. For labeling with technetium-99 m, the hydrazino-modified protein is reacted with a reduced technetium species, formed by reacting pertechnetate with a reducing agent in the presence of a chelating dioxygen ligand. The technetium becomes bound to the protein through what are believed to be hydrazido or diazenido linkages with the coordination sphere completed by the ancillary dioxygen ligands. Examples of ancillary dioxygen ligands include glucoheptonate, gluconate, 2-hydroxyisobutyrate, and lactate.
In one embodiment of the invention described in Schwartz et. al., the bifunctional aromatic hydrazine or hydrazide is protected as a lower alkyl hydrazone. This was done to prevent cross-reaction between the hydrazine or hydrazide and the protein reactive substituent because in the absence of the protecting group the bifunctional compound reacts with a protein to form a hydrazone modified protein. The free hydrazine or hydrazide group on the protein is then formed by dialysis into an acidic (pH 5.6) buffer and mixed with a suitable metal species, such as a reduced technetium species, in acidic media to yield a labeled protein.
Although the lower alkyl hydrazone protecting group prevents the cross-reaction between the hydrazine or hydrazide and the protein reactive substituent, it can be displaced by other aldehydes and ketones to form different hydrazones. This is a serious and significant disadvantage. The presence of other aldehydes and ketones in small quantities is unavoidable in a commercial pharmaceutical manufacturing setting, because they are extracted from various plastic and rubber materials and are also used in common disinfectants. Small quantities of formaldehyde are particularly ubiquitous. Therefore, reagents comprised of lower alkyl hydrazone protected biologically active molecules can degrade into a number of different hydrazone containing species, depending on the number and quantities of other aldehydes and ketones to which they are exposed during processing or manufacturing or storage after manufacture. This presents a significant problem in maintaining the purity of the reagents, and thus renders lower alkyl protected reagents unattractive as commercial candidates.
The present invention provides novel reagents for the preparation of radiopharmaceuticals comprised of stable hydrazone modified biologically active molecules. The stable hydrazones do not react appreciably with other aldehydes and ketones, maintaining the purity of the reagents during manufacturing. Surprisingly, these stable hydrazone reagents are still reactive enough to be labeled with radionuclides such as technetium-99 m.